Project Summary/Abstract Background: Proteins control critical regulatory pathways in mammalian cell nuclei that underlie normal physiology and disease. Some proteins recognize chromosomal DNA to regulate transcription while others bind to pre-mRNA to alter splicing. Proteins, however, lack a general capacity to be highly selective for recognition of just one gene and or rapidly evolve selectivity for new genes. We hypothesize that RNA can form networks on chromatin that act to complement protein transcription and splicing factors. Long noncoding RNAs (lncRNAs) overlap the 3' and 5' termini of most mRNAs. As lncRNAs are synthesized they are in close proximity to their related protein-encoding genes. Unlike sequences within chromosomal DNA, lncRNAs display sequences in a form that is readily accessible to sequence-specific base-pairing by small RNAs and subsequent action to affect gene expression. Objective: Our long-term goal is to understand the potential for RNA to interact with chromatin to regulate transcription or splicing. We will pay specific attention to argonaute (AGO) protein because of its key role in promoting RNA-RNA association. Aim 1. Mechanism of Nuclear AGO. While RNAi is well studied in the cytoplasm almost nothing is known about mechanisms for recognition in the nucleus. We will examine subcellular localization, strand loading, and the potential for stand cleavage in nuclear extracts and on chromatin. These data will reveal how AGO functions in the nucleus to control gene expression. Aim 2. Identify and Validate Action of Proteins Associated with Nuclear AGO. Understanding interactions between AGO and other proteins is essential for comprehending function in the nucleus. We will use mass spectrometry to identify protein partners for AGO and build a detailed mechanistic model of nuclear control networks. Aim 3. Identify RNAs Associated with Nuclear AGO. Nuclear AGO has the potential to be a critical organizer of RNA control networks in cell nuclei. We will use RNA immunoprecipitation with anti-AGO antibodies followed by high throughput deep sequencing as an unbiased approach to identify RNA sequences in nuclei that interact with AGO. We will examine associations between AGO and miRNAs, mRNA, and long noncoding transcripts and predict endogenous RNA interactions on chromatin. Positive impacts include: 1) Improved understanding for how AGO and small RNAs promotes sequence- specific recognition of noncoding RNAs in cell nuclei and on chromatin; 2) Identification of RNA partners for AGO, information critical for understanding how RNA recognition leads to altered splicing of transcription; and 3) An appreciation of endogenous RNA-mediated regulatory pathways in cell nuclei and the potential of small RNAs to control gene expression for therapeutic or biotechnological applications.